1. Field of the Invention
This invention relates to liposomes and in particular to an extrusion technique for the rapid production of unilamellar liposomes.
2. Description of the Prior Art
As is well known in the art, liposomes are closed vesicles having a lipid bilayer membrane surrounding an aqueous core. In general, liposomes of the following three types have been produced: (1) multilamellar vesicles (MLVs) wherein each vesicle includes multiple concentric bilayer membranes stacked one inside the other in an onion skin arrangement: (2) small unilamellar vesicles (SUVs) having only one bilayer membrane per vesicle and having diameters ranging up to about 50 nm; and (3) large unilamellar vesicles (LUVs), again having only one bilayer membrane per vesicle, but in this case having diameters greater than about 50 nm and typically on the order of 100 nm and above.
A review of these three types of liposomes, including methods for their preparation and various uses for the finished liposomes, can be found in the text Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, the pertinent portions of which are incorporated herein by reference. See also Szoka, Jr., et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980), the pertinent portions of which are also incorporated herein by reference.
Other types of liposomes which have been developed include stable plurilamellar vesicles (SPLVs), monophasic vesicles (MPVs), and steroidal liposomes. Descriptions of these vesicles and methods for preparing them can be found in copending and commonly assigned U.S. patent applications Ser. Nos. 476,496, 521,176, and 599,691, filed on Mar. 24, 1983, and now U.S. Pat. No. 4,522,803 Aug. 8, 1983, and now U.S. Pat. No. 4,588,578 and Apr. 12, 1984, and now abandoned respectively, the pertinent portions of which are incorporated herein by reference.
The present invention relates to an improved method for the production of liposomes. In particular, it relates to an improved method for producing unilamellar liposomes of both the large and small types.
Prior to the present invention, large unilamellar liposomes (LUVs) were commonly produced by one of the following three methods: (1) reverse-phase evaporation, (2) detergent dilution, and (3) infusion procedures using various solvents. See Liposomes, supra, Ch. 1, pages 37-44.
In the reverse-phase evaporation technique, an aqueous buffer is introduced into a mixture of phospholipid and an organic solvent to produce "inverted micelles," i.e., droplets of water stabilized in the organic solvent by being surrounded by a phospholipid monolayer. Evaporation of the solvent causes the micelles to coalesce and form the desired liposomes. See, for example, Szoka, Jr., et al., Proc. Natl. Acad. Sci. USA, 75:4194 (1978); and U.S. Pat. No. 4,235,871 to Papahadjopoulos et al.
In the detergent dilution approach, lipid, detergent and an aqueous solution are mixed together and sonicated to form the desired vesicles. Separation techniques, such as, gel filtration, are then used to remove the detergent and thus produce the finished liposomes.
In the infusion procedures, lipid is dissolved in a solvent, e.g., pentane or diethyl ether, and the lipid-solvent solution is infused into an aqueous solution under conditions that cause the solvent to vaporize and thus produce the desired liposomes. See, for example, Deamer, Annals New York Academy of Sciences, 308:250-258 (1978).
Other techniques which have been used to produce LUVs include fusion techniques whereby a population of SUVs is treated so as to cause individual SUVs to fuse with each other to form LUVs. For example, U.S. Pat. No. 4,078,052 to P. Demetrios Papahadjopoulos describes a technique wherein calcium ions are used to fuse SUVs into cochleate cylinders, and the cylinders are then treated with a calcium chelating agent such as EDTA to form the desired LUVs. Rapid freezing of SUVs, followed by slow thawing, has also been used to produce LUVs by fusion. See, for example, U. Pick, Archives of Biochemistry and Biophysics, 212:186 (1981).
With regard to the production of SUVs, as with LUVs, a variety of techniques have been employed in the past. See Liposomes, supra, Ch. 1, pages 33, 36. The earliest technique involved sonication to clarity of a suspension of lipid in an aqueous solution using a probe or bath sonication unit. Other techniques have included infusion procedures along the lines of those used for producing LUVs but with ethanol as the solvent (see S. Batzri and E. Korn, Biochimica et Biophysica Acta, 298:1015 (1973)), and a technique employing multiple passes of MLVs through a French press operated at a pressure of 20,000 psi (see, for example, Hamilton, Jr., et al., Journal of Lipid Research, 21:981 (1980); and Barenholz, et. al., FEBS Lett., 99:210 (1979)).
In addition to the basic techniques used to produce liposomes, various ancillary techniques have been developed for post-preparation treatment of liposomes to improve their properties. In particular, many of the LUV techniques described above have required sizing of the finished liposomes by filtration using, for example, a series of polycarbonate filters. See Liposomes, supra, Ch. 1, pages 37-39, 45: and Szoka, et al., Biochimica et Biophysica Acta, 601:559 (1980). Series of polycarbonate filters have also been used to size MLVs. See F. Olson, et al., Biochimica et Biophysica Acta, 557:9 (1979), and Bosworth, et al., Journal of Pharmaceutical Sciences, 71:806 (1982).
Although each of the foregoing techniques can be used to produce liposomes, none of these techniques are totally satisfactory. For example, each of the commonly used LUV techniques involves combining the components making up the liposome with a lipid solubilizing agent, i.e., either an organic solvent or a detergent. As is well known in the art, solvents and detergents can adversely effect many materials, such as enzymes, which one may want to encapsulate in liposomes, and thus these techniques cannot be used with these materials. Also, in applications such as the generation of drug carrier systems, the possible presence of these potentially toxic agents is undesirable.
Moreover, these techniques often require lengthy dialysis procedures which can never completely remove the solvent or detergent employed. See, for example, T. Allen, et al., Biochimica et Biophysica Acta, 601:328 (1980). Further, a variety of protocols are required depending on the lipid species. For example, the limited solubility of certain lipids (e.g., cholesterol, phosphatidylethanolamine (PE), and phosphatidylserine (PS)) in ether or ethanol requires modification of techniques employing these solvents. Alternatively, detergent dialysis procedures employing non-ionic detergents such as octylglucoside are tedious to apply as they can involve several days of dialysis. Plainly, the need to separate lipid solubilizing agents from the finished liposomes materially decreases the usefulness of these methods.
Along these same lines, the prior art LUV techniques have, in general, produced liposomes of various sizes, as well as aggregates of liposomes, thus requiring the additional step of sizing the finished liposomes with a series of filters. Again, this makes the overall process more time consuming and complicated.
The fusion techniques include similar drawbacks. For example, the calcium ion/calcium chelating agent technique, like the solvent and detergent techniques, involves the use and subsequent removal of materials in addition to those actually making up the finished liposomes, in this case, the chelating agent and the added calcium ions. As with the solvents and detergents, these materials represent possible sources of contamination, limit the usefulness of the technique, and make the technique more complicated. Also, this technique requires that the composition of the liposomes includes some phosphatidylserine.
As to the freeze-thaw technique, this technique suffers from the drawback that the specific trapping capacity of the liposomes produced by the technique drops off sharply at phospholipid concentrations above about 20 mg/ml.
The SUV techniques have similar problems. For example, high energy sonication can cause oxidation and degradation of phospholipids and may damage solute molecules which one wants to capture in the interior space of the liposomes. Also, when performed using a sonication probe, high energy sonication can cause probe erosion, and if done with bath sonication in combination with radioactive materials, can produce a potentially hazardous aerosol. Low energy sonication is slow, can be destructive to phospholipid molecules, and cannot be used to prepare large quantities of liposomes. Further, the sonication approach results in low trapping efficiencies.
The infusion type SUV procedures suffer the same problems as the LUV infusion procedures. The high pressure French press technique has its own set of problems, including difficulties in making the technique repeatable, the need for post-preparation filtration to remove those MLVs which have not been converted to SUVs, the need for expensive and cumbersome equipment capable of withstanding the high pressures used, and contamination of the product by disintegration of components of the apparatus which occurs during processing of the liposomes. See, for example, Bosworth, et al., Journal of Pharmaceutical Sciences, 71:806 (1982). Also, this technique can only produce small liposomes having a low trapping efficiency.